1. Field of the Invention
This invention relates to a novel method of conducting chemiluminescence-based assays. More particularly, this invention relates to chemiluminescence-based assays in an aqueous environment in which a first, light-producing, enzymatic reaction is carried out at a pH at which an enzyme is active and using as a substrate an enzyme-cleavable dioxetane derivative, and carrying out a second light-producing reaction under strongly alkaline conditions to generate visible light as a sustained glow of greater intensity than that generated in the first light-producing reaction.
2. Description of Prior Art
Chemical and biochemical assays in which the presence or concentration, or both, of an analyte is measured by luminescence assays are based on either chemiluminescent or bioluminescent reactions, and are commonly utilized as highly sensitive methods of ultramicroanalysis. Such prior art methods of chemiluminescence assay include: (1) H.sub.2 H.sub.2 measurement with luminol/potassium ferricyanide in the presence of an alkali [Bostick et al., Anal. Chem. 47:447-451 (1975)]; (2) glucose measurement with luminol-glucose oxidase [Bostick et al., 1975]; and (3) hemoglobin measurement with luminol/H.sub.2 H.sub.2 in the presence of an alkali [Ewetz, L. et al., Anal. Biochem., 71:564-570 (1976)]; (4) ATP measurement with Photinus pyralis luciferin-luciferase [Addanki et al., Anal. Biochem., 14:261-264 (1966)]; (5) measurement of intracellular free calcium ions with aequorin [Blinks et al., Pharmacol. Rev., 28:1-93 (1976)]; and (6) NADH measurement with bacterial luciferase [Hasting, J.W. et al., Ann. Rev. Microbiol., 31:549 (1977)].
These conventional methods of chemiluminescence assays described exhibit several problems. Method (1) is incapable of precise measurements; either the sample itself is decomposed by the oxidizer used to produce luminescence or the oxidizer itself is decomposed. Method (2) is difficult to carry out in aqueous systems; the reagents used to produce luminescence are poorly soluble in water and hence are not suitable for bioassays. Method (3) involves an intermittent luminescent reaction, and requires considerable skill in achieving timed measurements. Methods (4)-(6) require the use of more expensive enzymes than those used in the assays of the present invention, enzymes which are also inactivated to a large extent during immunoassays.
Previous methods of detecting a substance using enzymatically-induced decomposition of enzyme cleavable dioxetane derivatives [Bronstein, WO 88/00695, published Jan. 28, 1988; Schaap, A.P., et al., Tetrahedron Lett , 28:1155 (1982)]have been conducted at pH values which were most suitable for enzyme activity. Although the sensitivities of biochemical assays based upon the aforementioned principle were much greater and more efficient than those previously obtained with the luminescence-based assays such as those cited above, the sensitivities were limited to those produced at the pH values optimal for enzyme activity.